IL-13Rα2 Expression Research Articles

Interleukin-13 receptor alpha 2 as a marker of poorer prognosis in high-grade astrocytomas.

Interleukin-13 receptor alpha 2 (IL13Rα2) is considered a prognostic marker for gliomas according to the World Health Organization (WHO) grade. However, the expression levels of the marker vary from case to case, even within the same grade. We investigated whether IL13Rα2 could serve as a predictor of poorer prognosis in gliomas. mRNA expression of IL13Rα2 was measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 52 formalin-fixed paraffin-embedded glioma samples (4 pilocytic astrocytomas, 9 diffuse astrocytomas, 12 anaplastic astrocytomas, and 27 glioblastomas, grade IV). The expression levels were compared with regard to WHO grade, MIB-1 index, patient's age, and overall survival. Real time qRT-PCR showed that IL13Rα2 is expressed in a subset of cases with a progressive increase from low- to high-grade astrocytomas (HGAs). The expression had a significant positive correlation with the MIB-1 index and advanced patient age at diagnosis. The overall survival (OS) of patients who had HGAs with higher levels of IL13Rα2 expression was significantly lower than the OS of those with HGAs with lower levels of IL13Rα2. In the 39 HGA cases studied, the median survival benefit in the lower expression group was 167.4 months. The median OS (mOS) in HGA group with lower IL13Rα2 expression was 186.4 months, while the mOS in the group with higher IL13Rα2 expression was 18.6 months (P=0.033). The hazard ratio, adjusted by fitting the Cox proportional hazard models for the mOS in the HGAs with higher IL13Rα2 levels and the HGGs with lower IL13Rα2 levels, was 5.97 (95% CI: 1.76 to 20.32). The results showed that IL13Rα2 may be used as a marker of poorer prognosis in HGAs, even among tumors of the same grade.

Disruption of the TWEAK/Fn14 pathway prevents 5-fluorouracil-induced diarrhea in mice.

AIMTo clarify the roles of TWEAK and its receptor Fn14 in 5-fluorouracil (5-FU)-induced diarrhea.METHODSDiarrhea was induced in wild-type (WT), Fn14 knockout (KO), and IL-13 receptor (IL-13R)α1 KO BALB/c mice using a single injection of 5-FU. Histological analysis, cytokine analysis, and flow cytometry was performed on ileal tissues and cells. Murine colon carcinoma-bearing mice were co-treated with an anti-TWEAK antibody and 5-FU. Embryonic fibroblast response to cytokines was also analyzed.RESULTS5-FU induced high Fn14 expression in epithelial cells. The severity of 5-FU-induced diarrhea was lower in Fn14 KO mice compared with WT mice. Administration of anti-TWEAK antibody reduced 5-FU-induced diarrhea without affecting the antitumor effects of 5-FU in vivo. 5-FU-induced expression of IL-13, IL-17A, TNF-α, and IFN-γ in the ileum was Fn14 dependent. The severity of 5-FU-induced diarrhea was lower in IL-13Rα1 KO mice, indicating major role for IL-13 signaling via IL-13Rα1 in pathogenesis. We found that IL-13Rα2, an IL-13 neutralizing/cell protective receptor, was strongly induced by IL-33 in vitro and in vivo. IL-13Rα2 was upregulated in the ileum of 5-FU-treated Fn14 KO mice. Thus, the deletion of Fn14 upregulated IL-13Rα2 expression, which reduced IL-13 expression and activity.CONCLUSIONDisruption of the TWEAK/Fn14 pathway affects several interconnected pathways, including those associated with IL-13, IL-33, and IL-13Rα2, to attenuate 5-FU-induced intestinal side effects.

Abstract 1498: Development of an IL13Ralpha2 x CD3 bispecific DART® protein for redirected T-cell killing of solid tumors

Abstract Introduction. IL13Rα2 is a membrane-bound protein expressed on a number of malignant tumors, but not at significant levels in most normal tissues. Clinically tested therapies to treat cancer by targeting IL13Rα2 to date have included a ligand toxin conjugate (IL13-PE) and IL13Rα2 directed CAR-T cells. Both strategies appear to be well tolerated, although with mixed results in tumor eradication. An alternative strategy for targeting IL13Rα2 is an IL13Rα2 x CD3 Dual-Affinity Re-Targeting (DART®) bispecific molecule designed to co-engage IL13Rα2 on tumor cells and cytotoxic T cells through CD3, resulting in killing of tumor cells. Methods. Mouse monoclonal antibodies (mAbs) were generated using standard immunization protocols followed by binding and epitope binning analyses. Cell binding was performed by flow cytometry and frozen normal and tumor tissues analyses by immunohistochemistry (IHC). In vitro studies were performed with cancer cell lines and primary human T cells or peripheral blood mononuclear cells (PBMCs). In vivo studies were performed in immune-deficient tumor-bearing mice co-implanted with activated human T cells or reconstituted with human PBMCs. Results. A panel of mAbs that bind human and cynomolgus monkey IL13Rα2 proteins were selected, representing a range of binding characteristics and epitope diversity. IHC showed favorable normal vs tumor tissue reactivity, with high levels of IL13Rα2 expression in glioblastoma and melanoma. The mAb panel was converted to DART molecules with an anti-CD3 arm, assessed for the ability to mediate cytotoxic T lymphocyte (CTL) activity and cytokine release and a lead selected for humanization and engineering into an IL13Rα2 x CD3 DART molecule incorporating an Fc domain to prolong circulating half-life. The IL13Rα2 x CD3 DART protein bound human and cynomolgus IL13Rα2 (KD = 0.64 nM and 0.5 nM, respectively) and mediated CTL activity against LOX-IMVI (melanoma) and SK-MES-1 (lung adenocarcinoma) target cells. Cytokines (IFN-γ, IL-10, and TNF-α) were released in the presence of IL13Rα2-expressing target cells and human PBMC, but not with PBMCs alone. Administration of the IL13Rα2 x CD3 DART molecule (≤50 μg/kg IV for 4 consecutive days) prevented tumor growth when tumor cells (A375 and LOX-IMVI melanoma, U87 glioblastoma, or DAOY medulloblastoma cell lines) were co-mixed with activated human T cells and implanted subcutaneously in NOD/SCID/IL2gamma-chain null mice. Tumor eradication was also observed in NOD/SCID/IL2gamma-chain/MHCI KO mice reconstituted with human PBMCs and bearing established LOX-IMVI melanoma tumors following 2 weekly IV doses of 50 μg/kg IL13Rα2 x CD3 DART molecule. Conclusions: Robust in vitro and in vivo antitumor activity for an IL13Rα2 x CD3 DART protein was demonstrated. Additional studies are underway to further characterize the molecule as a development candidate for treatment of IL13Rα2-positive cancers. Citation Format: Jill Rillema, Monica Licea, Shereen Saini-Lal, Doug Smith, Francine Chen, Annie Lam, Yinhua Yang, Liqin Liu, James Tamura, Ralph Alderson, Ezio Bonvini, Syd Johnson, Paul Moore. Development of an IL13Ralpha2 x CD3 bispecific DART® protein for redirected T-cell killing of solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1498.

76. Transgenic Expression of IL15 Improves Antiglioma Activity of IL13Rα2-CAR T Cells

BACKGROUND: Glioblastoma (GBM) is the most aggressive primary brain tumor in humans, and is virtually incurable with conventional therapies. Immunotherapy with T cells expressing chimeric antigen receptors (CARs) specific for the GBM antigen IL13Rα2 is an attractive approach to improve outcomes. We recently generated the first scFv-based CAR that is specific for IL13Rα2 and demonstrated that IL13Rα2-CARs with a CD28.ζ endodomain had potent anti-GBM activity in preclinical models. However, CAR T-cell persistence was limited, resulting in recurrence of IL13Rα2-positive GBMs. Since IL13Rα2-CARs with other endodomains (41BB, 41BB and OX40) did not improve outcomes, the goal of this project was to evaluate if transgenic expression of IL-15, a cytokine that is critical for T-cell proliferation and survival, enhances persistence and anti-tumor activity of IL13Rα2-CAR.CD28.ζ T cells.METHODS: We generated IL13Rα2-CAR.CD28.ζ T cells expressing IL-15 (IL13Rα2-CAR. IL15 T cells) by double transducing T cells with retroviruses containing expression cassettes encoding i) IL13Rα2-CAR.CD28.ζ or ii) IL-15, ΔNGFR, and iC9 separated by 2A sequences. We determined the effector function of IL13Rα2-CAR. IL15 T cells in vitro using standard assays, and in the U373 GBM xenograft model.RESULTS: Double transduction of CD3/CD28-activated T cells resulted in T-cell lines that expressed both transgenes in 45-50% of T cells. At base line IL13Rα2-CAR. IL15 T cells produced on average 69.5 pg/ml of IL15. Production was significantly increased after CD3 or antigen-specific T-cell stimulation (176.7 pg/ml; n=6; p<0.001). IL13Rα2-CAR. IL15 T cells were as efficient as IL13Rα2-CAR T cells in killing IL13Rα2-positive GBMs in vitro. After intratumoral injection into U373 glioma-bearing mice IL13Rα2-CAR. IL15 T cells persisted significantly longer than IL13Rα2-CAR T cells (p<0.05). This resulted in a significant increase in progression free (98 vs 49 days; p=0.004) and overall survival (p=0.006) of treated mice. Up to date, 4/10 IL13Rα2-CAR. IL15 T-cell treated mice remain glioma free with a follow up of at least 80 days. Recurring U373 gliomas post IL13Rα2-CAR. IL15 T-cell therapy had down regulated IL13Rα2 expression, indicating immune escape. This was specific for the targeted antigen since LL13Rα2-CAR.LL15 T-cell-treated U373 gliomas continued to express other tumor antigens such as EphA2 and HER2 at unchanged levels in comparison to controls.CONCLUSION: Here we demonstrate that transgenic expression of IL15 enhances the in vivo persistence of IL13Rα2-CAR T cells resulting in improved anti-glioma activity. However, enhanced in vivo persistence of T cells also resulted in the development of antigen-loss variants highlighting the need to target multiple antigens in tumors with heterogeneous antigen expression such as GBM.

Sputum Gene Expression of IL-13 Receptor α2 Chain Correlates with Airflow Obstruction and Helper T-Cell Type 2 Inflammation in Asthma.

BRP-39/YKL-40 is a chitinase-like protein that plays a critical role in IL-13-induced inflammation. It correlates positively with asthma severity and airway remodeling ( 1 ) via binding to IL-13 receptor α2 chain (IL-13Rα2). Because the relationship of IL-13Rα2 to human asthma has never been evaluated previously, we sought to determine the relationship between IL-13Rα2, YKL-40, and asthma. We evaluated 112 patients (69% women) with a mean age of 46.7 years. Subjects completed an asthma phenotyping protocol that included analysis of sputum gene expression by Affymetrix 1.0 ST gene array (Affymetrix, Santa Clara, CA) and YKL-40 protein levels. IL-13Rα2 gene expression was readily detectable in the sputum and correlated negatively with prebronchodilator (BD) FEV1 (rs = -0.282, P < 0.01), post-BD FEV1 (rs = -0.268, P < 0.01), pre-BD FEV1/FVC ratio (rs = -0.228, P < 0.05), and post-BD FEV1/FVC ratio (rs = -0.242, P < 0.01). IL-13Rα2 gene expression correlated positively with gene expression of IL-13 (rs = 0.484, P < 0.001), IL-5 (rs = 0.237, P < 0.05), and IL-8 (rs = 0.218, P < 0.05). Regression analysis showed that the post-BD FEV1/FVC ratio is significantly associated with IL-13Rα2 expression and CHI3L1 expression in sputum after controlling for IL-4, IL-5, IL-13, and transforming growth factor-β1 gene expression (all P < 0.01). Sputum YKL-40 gene expression positively correlated with IL-8 expression (rs = 0.357, P < 0.001) and negatively correlated with pre- and post-BD FEV1/FVC ratios (rs = -0.299, P < 0.001 and rs = -0.305, P < 0.01, respectively). Sputum and serum YKL-40 protein levels were not associated with IL-13Rα2 expression. This analysis demonstrates that IL-13Rα2 is associated with reduced lung function, helper T-cell type 2 gene expression, and airflow obstruction in the airway of individuals with asthma, which might in turn be driven by airway remodeling. Future studies will be required to define the proinflammatory and remodeling effects of this receptor that up to now has been considered solely a modulator of IL-13-induced inflammation.

IL13Rα2 signaling in colorectal cancer.

IL13 receptor α2 (IL13Rα2) is a high-affinity IL13 receptor overexpressed in a variety of human cancer types. In many of these cancers (glioblastoma, head and neck squamous cell carcinoma and ovarian cancer) the expression of this receptor is associated with poor prognosis [1–3]. In colorectal cancer cells, IL13 signaling through its receptor IL13Rα2 plays a critical role in cell adhesion, invasion and liver metastasis. Moreover, overexpression of IL13Rα2 in human colon cancer samples was associated to metastasis and poor outcome of patients [4]. Because of the short cytoplasmic tail of IL13Rα2 (14 amino acids), it was initially regarded as a decoy, unable to transduce cell signaling. Recently, several articles described that the binding of IL13 to IL13Rα2 triggered the activation of several proteins, such as Src, AKT and AP-1 [4, 5]. By a proteomic approach using colon cancer cells, we identified the proteins that co-immunoprecipitated with IL13Rα2 and found a scaffold protein, FAM120A, also known as C9orf10 or OSSA [6], as a major interactor. FAM120A was overexpressed in colorectal cancer cell lines and 55% of human colon cancer specimens. Still, FAM120A was a quite uncharacterized protein, with no catalytic activity, which was described either as a promoter of Src activation by oxidative stress or a component of RNA-containing structures. Therefore, we investigated the protein network of FAM120A by quantitative proteomics. Proteins associated with FAM120A were involved in three cell membrane complexes: TRAIL receptor, G protein coupled receptor (GPR56) and matrix-cell adhesion networks. Many of the proteins belonging to these complexes were signaling proteins, suggesting that FAM120A facilitates the interaction and activation of such proteins after different extracellular stimuli. In fact, IL13 triggered the recruitment into the IL13Rα2/FAM120A complex and the activation of several signaling molecules, such as FAK, Src, PI3K, AKT, mTOR and ERK1/2. FAM120A was required for the activation of PI3K/AKT/mTOR pathway by Src kinases and for the activation of FAK. Moreover, we found that FAM120A was associated to proteins involved in vesicle trafficking, suggesting a role in protein transport that is common in scaffold proteins. In fact, the silencing of FAM120A provoked a partial reduction in IL13Rα2 expression in the cell membrane, whereas total IL13Rα2 expression remained unchanged. Moreover, FAM120A silencing caused a significant downregulation of other scaffold proteins and linkers to cytoskeleton, such as AHNAK, SH3PXD2B, SPTAN1, PDLIM7 and DSG2 as well as some proteins involved in protein transport such as MYO1D, S100A10 and ACTN4. Finally, FAM120A silencing down-regulated three proteins involved in RNA splicing (NUDT21, SFPQ and SF382), which may explain the previous link between FAM120A and RNA biology. In summary, FAM120A is a scaffold protein required for the proper IL13Rα2-triggered signaling, which is involved in colon cancer metastasis. FAM120A also modulates IL13Rα2 cell membrane location and promotes the expression of other scaffold proteins involved in signaling or transport. These results support the use of FAM120A as potential target for therapy and reinforce the therapeutic value of IL13Rα2 in colon cancer. In other cancer types, where IL13Rα2 has been described as a poor prognosis biomarker, FAM120A could also play an important role as regulator of IL13-triggered signaling. In fact, according to cBioPortal [7], FAM120A is overexpressed in sarcoma (28%), head and neck squamous cell carcinoma (24%), adrenocortical carcinoma (20%), pancreatic adenocarcinoma (12%) and brain glioma (12%). Furthermore, a role for IL13Rα2 has been shown for diverse pathologies, as asthma or ulcerative colitis. FAM120A is associated to other complexes and may play a role in the signaling after other stimuli besides IL13. We found FAM120A related with focal adhesion proteins where may collaborate in FAK activation. Also, FAM120A may be involved in GPR56 signal transduction, as this receptor for type III collagen activates RhoA, and FAM120A is associated to RhoGEFs. Finally, in some cancer cell types proapoptotic TRAIL receptor is actually promoting survival by activation of different kinases, including PI3K. FAM120A is associated to a TRAIL receptor and promotes PI3K activation; thus, it may have a potential role in anti-apoptotic conversion of the TRAIL receptor. Therefore, the role of IL13Rα2/FAM120A in different pathologies open new research lines with important implications in asthma or ulcerative colitis.

GdCl3 Attenuates Schistosomiasis japonicum Egg-Induced Granulomatosis Accompanied by Decreased Macrophage Infiltration in Murine Liver.

Early-stage hepatic granuloma and advanced-stage fibrosis are important characteristics of schistosomiasis. The direct consequences of gadolinium chloride (GdCl3) in egg-induced granuloma formation have not been reported, although GdCl3 is known to block the macrophages. In present study, mice were infected with 15 Schistosoma japonicum (S. japonicum) cercariae and treated with GdCl3 (10 mg/kg body weight) twice weekly from day 21 to day 42 post-infection during the onset of egg-laying towards early granuloma formation. Histochemical staining showed that repeated injection of GdCl3 decreased macrophages infiltration in liver of mice infected with S. japonicum. Macrophage depletion by GdCl3 during the initial phase attenuated liver pathological injury characterized by smaller granuloma size and decreased immune inflammation as well as less fibrogenesis. In addition, IL-13Rα2 expression was reduced by GdCl3 in liver of mice infected with S. japonicum. The results suggest that GdCl3 depleted macrophages, which attenuated helminth infected immune responses involving with IL-13Rα2 signal. These findings would highlight a therapeutic potential via manipulating IL-13Rα2+ macrophage in schistosomiasis.

IT-20 * OVEREXPRESSION OF IL13Ra2: ASSESSMENT OF PRO-AGGRESSIVE AND PRO-INVASIVE PHENOTYPES OF MALIGNANT GLIOMA IN A SYNGENEIC ANIMAL MODEL

BACKGROUND: IL13Rα2 is a promising target for novel therapies in glioblastoma (GBM) due to its overexpression in tumor cells as well as glioma-initiating cells, which are responsible for the growth and recurrence of GBM. The role of IL13Ra2, found heterogeneously expressed in GBMs, is not fully understood and better targeting strategies are necessary for its clinical application. This study investigated the effectiveness of different patterns of IL13Rα2 expression on the survival of syngeneic rodent glioma models. This study also aims to act as a blueprint for further tests of the level of immune response to the target immunotherapy in immunocompetent mice bearing GBMs with different levels of IL13Rα2 expression. METHODS: G26 murine glioma cell line, with transfected to express hIL13Rα2 (G26-H2) and vector control (G26-V2) were intracranially injected in two groups of C57Bl/6 mice respectively. The metric analyzed for outcome was survival. Histological analysis were used to evaluate the level of IL13Ra2 expression in primary tissue obtained from patients with high gliomas. RESULTS: Two populations of G26 glioma cell lines were established per hIL13Rα2 expression: the hIL13Rα2+ and the hIL13Rα2−. In vivo, the cell lines showed a significant difference in survival: the group that received intracranial injection of G26-H2 cells (100% positive for hIL13Rα2) had a median survival of 29.5 days compared to 49 days for group receiving G26-V2 cells (100% negative for h IL13Rα2), p <0.0172. CONCLUSION: We assessed two different populations of G26 glioma cell lines: h IL13Rα2+ and h IL13Rα2−. In vivo, after intracranial implantation, the two populations showed a significant difference in survival. These results confirm the role of hIL13Rα2 overexpression in specific subtypes of GBMs, and most likely the most aggressive ones.

TNF-α/IL-17 synergy inhibits IL-13 bioactivity via IL-13Rα2 induction

TNF-α/IL-17 synergy inhibits IL-13 bioactivity via IL-13Rα2 induction

Cytokine induction of VCAM-1 but not IL13Rα2 on glioma cells: a tale of two antibodies.

The interleukin-13 receptor alpha2 (IL13Rα2) is a cell surface receptor that is over-expressed by a subset of high-grade gliomas, but not expressed at significant levels by normal brain tissue. For both malignant and non-malignant cells, IL13Rα2 surface expression is reported to be induced by various cytokines such as IL-4 or IL-13 and tumor necrosis factor (TNF). Our group has developed a therapeutic platform to target IL13Rα2-positive brain tumors by engineering human cytotoxic T lymphocytes (CTLs) to express the IL13-zetakine chimeric antigen receptor. We therefore sought to investigate the potential of cytokine stimulation to induce IL13Rα2 cell surface expression, and thereby increase susceptibility to IL13Rα2-specific T cell killing. In the course of these experiments, we unexpectedly found that the commercially available putative IL13Rα2-specific monoclonal antibody B-D13 recognizes cytokine-induced VCAM-1 on glioblastoma. We provide evidence that the induced receptor is not IL13Rα2, because its expression does not consistently correlate with IL13Rα2 mRNA levels, it does not bind IL-13, and it is not recognized by IL13-zetakine CTL. Instead we demonstrate by immunoprecipitation experiments and mass spectrometry that the antigen recognized by the B-D13 antibody following cytokine stimulation is VCAM-1, and that VCAM-1, but not IL13Rα2, is induced on glioma cells by TNF alone or in combination with IL-13 or IL-4. Further evaluation of several commercial B-D13 antibodies revealed that B-D13 is bi-specific, recognizing both IL13Rα2 and VCAM-1. This binding is non-overlapping based on soluble receptor competition experiments, and mass spectrometry identifies two distinct heavy and light chain species, providing evidence that the B-D13 reagent is di-clonal. PE-conjugation of the B-D13 antibody appears to disrupt IL13Rα2 recognition, while maintaining VCAM-1 specificity. While this work calls into question previous studies that have used the B-D13 antibody to assess IL13Rα2 expression, it also suggests that TNF may have significant effects on glioma biology by up-regulating VCAM-1.

IL-13Rα2-bearing, type II NKT cells reactive to sulfatide self-antigen populate the mucosa of ulcerative colitis

ObjectivePrevious studies have shown that ulcerative colitis (UC) is associated with the presence of lamina propria non-invariant (Type II) NKT cells producing IL-13 and mediating epithelial cell cytotoxicity. Here we...

Histone deacetylase inhibitiors enhance sensitivity of murine sarcoma tumors to IL-13-PE immunotoxin-based cancer therapy by upregulating IL-13Rα2 expression in vitro and in vivo

Interleukin-13 Receptor alpha 2 (IL-13Rα2) is a tumor antigen and a potent target for cancer therapy. Previously, we have reported that histone deacetylase (HDAC) inhibitors upregulated the IL-13Rα2 expression and enhanced anti-cancer effects of IL-13-PE, an immunotoxin composed of interleukin-13 and truncated Pseudomonas exotoxin, in a mouse model of human pancreatic cancer. Herein, we have investigated whether HDAC inhibitors can enhance the effect of IL-13-PE in immunocompetent mouse tumor models by upregulating IL-13Rα2. We analyzed mRNA and protein levels of mouse IL-13Rα2 in four mouse tumor cell lines, MCA304 sarcoma, 4T1 breast cancer, GL261 glioma and D5 melanoma. By RT-PCR analysis, mRNA levels for IL-13Rα2 were high in MCA304, moderate in 4T1, low in GL261 and below the detection limit in D5 mouse tumor cell lines. These cell lines were treated with three types of HDAC inhibitors, trichostatin A, sodium butyrate and suberoylanilide hydroxamic acid (SAHA) to evaluate their effects on IL-13Rα2 expression in vitro. All HDAC inhibitors dramatically increased IL-13Rα2 mRNA expression in MCA304, 4T1, and GL261 cell lines. The D5 tumor cell line showed only a slight increase in mRNA levels of IL-13Rα2. Western blot analysis for IL-13Rα2 demonstrated increased levels of IL-13Rα2 protein in all of the HDAC inhibitor treated tumor cells. We also observed that all three HDAC inhibitors selectively enhanced cytotoxicity of IL-13-PE in MCA304 and 4T1 cell lines. TSA treatment resulted in maximum improvement of IL-13-PE induced cytotoxicity in MCA304, NaB in GL261, and SAHA in 4T1 cell lines. We next developed a subcutaneous tumor model of mouse sarcoma by implanting MCA304 tumor cells in C57BL/6 mice. Mice were treated with SAHA (an FDA licensed drug) i.p. at 25mg/kg/day dose from day 4 through 9 prior to IL-13-PE immunotoxin treatment. IL-13-PE was administered at a 250mg/kg dose intratumorally from day 5 through 9. Mice treated with vehicle control and SAHA showed tumor growth to ~1500 mm3 in ~22 days and were sacrificed because of ethical reasons. Treatment with IL-13-PE alone showed a significant reduction in tumor growth and mice survived longer. Tumor size reached ~750 mm3 in 35 days. However, when IL-13-PE was administered to SAHA pretreated mice, tumor size showed further reduction and reached only ~400 mm3 on day 35. Additional studies are ongoing to determine the mechanism of synergistic antitumor effects of HDAC inhibitors and IL-13-PE. These results suggest that HDAC inhibitors may upregulate tumor antigens in vivo and thus may be considered in combination with cancer vaccines and immunotherapy products for cancer therapy.

IL-13 signaling via IL-13Rα2 triggers TGF-β1-dependent allograft fibrosis

BackgroundAllograft fibrosis still remains a critical problem in transplantation, including heart transplantation. The IL-13/TGF-β1 interaction has previously been identified as a key pathway orchestrating fibrosis in different inflammatory immune disorders. Here we investigate if this pathway is also responsible for allograft fibrosis and if interference with the IL-13/TGF-β1 interaction prevents allograft fibrosis.MethodsFVB or control DBA/1 donor hearts were transplanted heterotopically into DBA/1 recipient mice and hearts were explanted at day 60 and 100 post-transplantation. Cardiac tissue was examined by Masson’s trichrome staining and immunohistochemistry for CD4, CD8, CD11b, IL-13, Fas ligand, matrix metalloproteinase (MMP)-1, MMP-13, β2-microglobulin, and Gremlin-1. Graft-infiltrating cells were isolated and analyzed by flow cytometry. IL-13 and TGF-β1 levels were determined by enzyme-linked immunosorbent assay (ELISA) and the amount of collagen was quantified using a Sircol assay; IL-13Rα2 expression was detected by Western blotting. In some experiments IL-13/ TGF-β1 signaling was blocked with specific IL-13Rα2 siRNA. Additionally, a PCR array of RNA isolated from the allografts was performed to analyze expression of multiple genes involved in fibrosis.ResultsBoth groups survived long-term (>100 days). The allogeneic grafts were infiltrated by significantly increased numbers of CD4+ (P <0.0001), CD8+ (P <0.0001), and CD11b+ cells (P = 0.0065) by day 100. Furthermore, elevated IL-13 levels (P = 0.0003) and numbers of infiltrating IL-13+ cells (P = 0.0037), together with an expression of IL-13Rα2, were detected only within allografts. The expression of IL-13 and IL-13Rα2 resulted in significantly increased TGF-β1 levels (P <0.0001), higher numbers of CD11bhighGr1intermediateTGF-β1+ cells, and elevated cardiac collagen deposition (P = 0.0094). The allograft fibrosis found in these experiments was accompanied by upregulation of multiple profibrotic genes, which was confirmed by immunohistochemical stainings of allograft tissue. Blockage of the IL-13/TGF-β1 interaction by IL-13Rα2 siRNA led to lower numbers of CD11bhighGr1intermediateTGF-β1+, CD4+, CD8+, and CD11b+ cells, and prevented collagen deposition (P = 0.0018) within these allografts.ConclusionsIL-13 signaling via IL-13Rα2 induces TGF-β1 and causes allograft fibrosis in a murine model of chronic transplant rejection. Blockage of this IL-13/TGF-β1 interaction by IL-13Rα2 siRNA prevents cardiac allograft fibrosis. Thus, IL-13Rα2 may be exploitable as a future target to reduce allograft fibrosis in organ transplantation.

Glioma IL13Rα2 Is Associated with Mesenchymal Signature Gene Expression and Poor Patient Prognosis

A major challenge for successful immunotherapy against glioma is the identification and characterization of validated targets. We have taken a bioinformatics approach towards understanding the biological context of IL-13 receptor α2 (IL13Rα2) expression in brain tumors, and its functional significance for patient survival. Querying multiple gene expression databases, we show that IL13Rα2 expression increases with glioma malignancy grade, and expression for high-grade tumors is bimodal, with approximately 58% of WHO grade IV gliomas over-expressing this receptor. By several measures, IL13Rα2 expression in patient samples and low-passage primary glioma lines most consistently correlates with the expression of signature genes defining mesenchymal subclass tumors and negatively correlates with proneural signature genes as defined by two studies. Positive associations were also noted with proliferative signature genes, whereas no consistent associations were found with either classical or neural signature genes. Probing the potential functional consequences of this mesenchymal association through IPA analysis suggests that IL13Rα2 expression is associated with activation of proinflammatory and immune pathways characteristic of mesenchymal subclass tumors. In addition, survival analyses indicate that IL13Rα2 over-expression is associated with poor patient prognosis, a single gene correlation ranking IL13Rα2 in the top ~1% of total gene expression probes with regard to survival association with WHO IV gliomas. This study better defines the functional consequences of IL13Rα2 expression by demonstrating association with mesenchymal signature gene expression and poor patient prognosis. It thus highlights the utility of IL13Rα2 as a therapeutic target, and helps define patient populations most likely to respond to immunotherapy in present and future clinical trials.

Abstract 2150: Therapeutic efficacy of an optimized quadruply mutated IL13-based bacterial cytotoxin in an orthotopic murine glioma model.

Abstract Interleukin 13 receptor alpha 2 (IL13Rα2) is a tumor-restricted plasma membrane receptor that is overexpressed on greater than 70% of Glioblastoma Multiformes (GBMs) as well as in other malignancies, including melanoma, adenocarcinoma, ovarian cancer and renal cell carcinoma. We and others have therapeutically exploited this attractive target using IL13-based targeting strategies to deliver bacterial toxins, chemotherapeutics, nanoparticles, viruses and immunotherapy directly to the IL13Rα2 tumor-restricted biomarker. In past work, we found multiple mutational hotspots on IL13 that (a) abrogated its binding to the physiologically abundant IL13Rα1/IL4Rα receptor heterodimer, and (b) increased its binding to the tumor-restricted IL13Rα2. Based on these findings, we created a delivery platform established as an optimized IL13Rα2-Targeted Quadruple Mutant of IL13 (TQM13; IL13.E13K/R66D/S69D/K105R) that we demonstrated binds with high affinity towards IL13Rα2, but not the IL13Rα1/IL4Rα receptor heterodimer. Of importance, we demonstrated the ability of radiolabeled TQM13 to target IL13Rα2-expressing tumors in vivo using positron emission tomography (PET). The objective of this current work was to demonstrate for the first time the in vivo IL13Rα2-targeted killing effect of our optimized TQM13 ligand genetically fused to a potent derivative of Pseudomonas exotoxin (PE). We successfully expressed TQM13 and TQM13-PE proteins in an E. coli expression system, denatured, refolded and purified them via Nickel-based affinity and ion exchange chromatography. We demonstrated IL13Rα2-specific affinity of TQM13 using a novel IL13Rα2-inducible system in which B16 melanoma cells expressed varying amounts of IL13Rα2 in proportion to the concentration of doxycycline. Of importance, the number of induced TQM13 binding sites (in vitro and in vivo) was comparable to the range seen in GBM and other malignancies (50,000-4,500,000 receptors/cell). In addition, we demonstrated that TQM13-PE killed cells in direct proportion to IL13Rα2 expression. Therefore, we tested the efficacy of TQM13-PE in an orthotopic murine GBM model by injecting mice with 1 μg of TQM13-PE intracranially on day 7 and 14 post stereotactic tumor cell implantation. We found a significant increase in survival only in mice bearing IL13Rα2-expressing orthotopic tumors, in contrast to mice bearing IL13Rα2-negative tumors. Thus, we have successfully generated an optimized, biomarker-targeted anti-GBM agent that demonstrated specific therapeutic potential against the tumor-associated IL13Rα2 biomarker in vivo. Citation Format: Van Nguyen, Dongqin Zhu, Jesse M. Conyers, Waldemar Debinski, Akiva Mintz. Therapeutic efficacy of an optimized quadruply mutated IL13-based bacterial cytotoxin in an orthotopic murine glioma model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2150. doi:10.1158/1538-7445.AM2013-2150

Targeting of interleukin-13 receptor α2 for treatment of head and neck squamous cell carcinoma induced by conditional deletion of TGF-β and PTEN signaling

BackgroundThe sixth leading class of cancer worldwide is head and neck cancer, which typically arise within the squamous epithelium of the oral mucosa. Human head and neck squamous cell carcinoma (HNSCC) is known to be difficult to treat and has only a 50% five-year survival rate. With HNSCC, novel therapeutics are needed along with a means of rapidly screening anti-cancer agents in vivo, such as mouse models.MethodsIn order to develop new animal models of cancer to test safety and efficacy of novel therapeutic agents for human HNSCC, tumors resembling clinical cases of human HNSCC were induced in the head and neck epithelium of a genetically engineered mouse model. This mouse model was generated by conditional deletion of two tumor suppressors, Transforming Growth Factor-β Receptor 1 (TGFβRI) and Phosphatase and Tensin homolog (PTEN), in the oral epithelium. We discovered that the tumors derived from these Tgfbr1/Pten double conditional knockout (2cKO) mice over-expressed IL-13Rα2, a high affinity receptor for IL-13 that can function as a tumor antigen. To demonstrate a proof-of-concept that targeted therapy against IL-13Rα2 expression would have any antitumor efficacy in this spontaneous tumor model, these mice were treated systemically with IL-13-PE, a recombinant immunotoxin consisting of IL-13 fused to the Pseudomonas exotoxin A.ResultsTgfbr1/Pten 2cKO mice when treated with IL-13-PE displayed significantly increased survival when compared to the untreated control mice. The untreated mice exhibited weight loss, particularly with the rapid onset of tongue tumors, but the treated mice gained weight while on IL-13-PE therapy and showed no clinical signs of toxicity due to the immunotoxin. Expression of IL-13Rα2 in tumors was significantly decreased with IL-13-PE treatment as compared to the controls and the number of myeloid-derived suppressor cells (MDSC) was also significantly reduced in the spleens of the IL-13-PE treated mice.ConclusionsOur study demonstrates that the Tgfbr1/Pten 2cKO mouse model of human HNSCC is a useful model for assessing antitumor activity of new cancer therapeutic agents, and that IL-13-PE has therapeutic potential to treat human head and neck cancer.

The innate antiviral response upregulates IL-13 receptor α2 in bronchial fibroblasts

IL-13 is key mediator of allergic inflammation in asthmatic patients. We have previously shown that the decoy receptor IL-13 receptor (IL-13R) α2 attenuates responses of fibroblasts to IL-13. Because the expression of IL-13Rα2 can be regulated by IFN-γ, a type II interferon, we hypothesized that innate antiviral responses characterized by type I interferon expression can also induce IL-13Rα2 expression. We sought to induce an innate antiviral response inprimary fibroblasts using exposure to double-stranded RNA(dsRNA) and to examine the expression and function of IL-13Rα2. Primary human fibroblasts were cultured from endobronchial biopsy specimens obtained from healthy or asthmatic volunteers and challenged with dsRNA. Upregulation of IL-13Rα2 mRNA was measured by using real-time quantitative PCR, and cell-surface IL-13Rα2 protein expression was measured by using fluorescence-activated cell sorting. Eotaxin release was determined by means of ELISA. Direct treatment with IFN-β led to an upregulation of IL-13Rα2. Exposure to dsRNA rapidly induced IFN-β mRNA in fibroblasts, and this was followed by significant induction of IL-13Rα2 mRNA and cell-surface protein expression, which was dependent on de novo protein synthesis. Aneutralizing antibody to the IFN-α/β receptor blocked cell-surface expression of IL-13Rα2 in the presence of dsRNA. Pretreatment of fibroblasts with dsRNA led to attenuation of IL-13-stimulated eotaxin production. However, the presence of an IL-13Rα2 neutralizing antibody restored IL-13-stimulated eotaxin production in dsRNA-treated cells. IFN-β induces IL-13Rα2 expression, leading to a consequential suppression of responsiveness to IL-13. These data suggest cross-talk between TH1 and TH2 pathways and point to an immunomodulatory role for IL-13Rα2 in human bronchial fibroblasts during viral infection.

MAPK Regulation of IL-4/IL-13 Receptors Contributes to the Synergistic Increase in CCL11/Eotaxin-1 in Response to TGF-β1 and IL-13 in Human Airway Fibroblasts

CCL11/eotaxin-1 is a potent eosinophilic CC chemokine expressed by primary human fibroblasts. The combination of TGF-β1 and IL-13 synergistically increases CCL11 expression, but the mechanisms behind the synergy are unclear. To address this, human airway fibroblast cultures from normal and asthmatic subjects were exposed to IL-13 alone or TGF-β1 plus IL-13. Transcriptional (nuclear run-on) and posttranscriptional (mRNA stability) assays confirmed that transcriptional regulation is critical for synergistic expression of CCL11. TGF-β1 plus IL-13 synergistically increased STAT-6 phosphorylation, nuclear translocation, and binding to the CCL11 promoter as compared with IL-13 alone. STAT-6 small interfering RNA significantly knocked down both STAT-6 mRNA expression and phosphorylation and inhibited CCL11 mRNA and protein expression. Regulation of the IL-4Rα complex by TGF-β1 augmented IL-13 signaling by dampening IL-13Rα2 expression, overcoming IL-13's autoregulation of its pathway and enhancing the expression of CCL11. Our data suggest that TGF-β1 induced activation of the MEK/ERK pathway reduces IL-13Rα2 expression induced by IL-13. Thus, TGF-β1, a pleiotropic cytokine upregulated in asthmatic airways, can augment eosinophilic inflammation by interfering with IL-13's negative feedback autoregulatory loop under MEK/ERK-dependent conditions.

High Expression of IL-13 Receptor α2 in Colorectal Cancer Is Associated with Invasion, Liver Metastasis, and Poor Prognosis

Autocrine secretion of cytokines by metastatic colorectal cancer cells and their role during invasion and liver homing has been poorly characterized. In this study, we used cytokine arrays to analyze the secretomes of poorly and highly metastatic colorectal cancer cells. Compared with poorly metastatic cancer cells, highly metastatic cells expressed increased levels of the immunosuppressive cytokines interleukin (IL)-4 and IL-13 in addition to increased surface expression of the high affinity IL-13 receptor IL-13Rα2, suggesting that IL-13Rα2 mediates IL-13 effects in colorectal cancer cells. Silencing of IL-13Rα2 in highly metastatic cells led to a decrease in adhesion capacity in vitro and a reduction in liver homing and increased survival in vivo, revealing a role for this receptor in cell adhesion, migration, invasion, and metastatic colonization. In support of this, IL-13 signaling activated the oncogenic signaling molecules phosphoinositide 3-kinase, AKT, and SRC in highly metastatic cells. Clinically, high expression of IL-13Rα2 was associated with later stages of disease progression and poor outcome in patients with colorectal cancer. Our findings therefore support a critical role for IL-13Rα2 expression in colon cancer invasion and metastasis.

Interleukin-13 receptor alpha2 is a novel therapeutic target for human adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a relatively rare but aggressive malignancy with limited therapeutic options. Previous genome-wide expression studies have demonstrated the overexpression of interleukin-13 receptor alpha2 (IL13Rα2) in some human malignancies. The authors evaluated IL13Rα2 mRNA and protein expression in 21 normal samples, 78 benign samples, 10 primary malignant samples, and 25 metastatic/recurrent samples and performed functional analyses with IL13 ligand and IL13 Rα2 knockdown in vitro. The sensitivity of 2 ACC cell lines (NCI-H295R [high IL13Rα2 expression] and SW13 [low IL13Rα2 expression]) to a highly specific IL-13 conjugated with Pseudomonas exotoxin (IL-13-PE) also was evaluated in both in vitro and in vivo models. IL13Rα2 was overexpressed in malignant tumors compared with benign and normal samples (15-fold higher; P < .05). Immunohistochemistry also confirmed higher protein expression in malignant and benign tumors than in normal adrenocortical tissues (P < .05). The half-maximal inhibitory concentration for IL-13-PE was 1.3 ng/mL in the NCI-H295R cell line and 1000 ng/mL in the SW13 cell line. Mice that received intratumoral or intraperitoneal IL-13-PE injection had a significant reduction in tumor size and increased tumor necrosis compared with control groups (P < .05) and also had prolonged survival (P < .05). IL13Rα2 protein expression increased in cells that were treated with IL-13 ligand along with cell invasion (P < .05). Direct IL13Rα2 knockdown decreased cellular proliferation and invasion (P < .05). The current results indicated that IL13Rα2 is overexpressed in ACC and regulates cell invasion and proliferation. IL13Rα2 is a novel therapeutic target for the treatment of human ACC.